Roseateles depolymerans gen. nov., sp. nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the beta-subclass of the Proteobacteria.

Abstract

Strains 61AT (T = type strain) and 61B2, the first bacteriochlorophyll (BChl) a-containing obligate aerobes to be classified in the beta-subclass of the Proteobacteria, were isolated from river water. The strains were originally isolated as degraders of poly(hexamethylene carbonate) (PHC). The organisms can utilize PHC and some other biodegradable plastics. The strains grow only under aerobic conditions. Good production of BChl a and caroterioid pigments is achieved on PHC agar plates and an equivalent production is observed under oligotrophic conditions on agar medium. Spectrometric results suggest that BChl a is present in light-harvesting complex I and the photochemical reaction centre. The main carotenoids are spirilloxanthin and its precursors. Analysis of the 16S rRNA gene sequence indicated that the phylogenetic positions of the two strains are similar to each other and that their closest relatives are the genera Rubrivivax, ideonella and Leptothrix with similarities of 96.3, 96.2 and 96.1%, respectively. The cells are motile, straight rods and contain poly-beta-hydroxybutyrate granules. Ubiquinone-8 is the predominant quinone. Vitamins are not required for growth. The G + C content of genomic DNA is 66.2-66.3 mol%. Genetic and phenotypic features suggest that the strains represent a new genus in the beta-subclass which is evenly distant from known genera. Consequently, the name Roseateles depolymerans gen. nov., sp. nov. is proposed for the strains; the type strain of Roseateles depolymerans is strain 61AT (= DSM 11813T).

Full text

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International Journal of Systematic Bacteriology (1 999), 49,449-457
Printed in Great Britain
Roseateles depolymerans gen. nov., sp. nov., a
new bacteriochlorophyll a-containing obligate
aerobe belonging to the P=subclass of the
Pro teobacteria
Tetsushi Suyama,’ Toru Shigematsu,’ Shinichi Takaichit2
Yoshinobu Nodasakaf3 Seizo F~jikawa,~
Yutaka Tokiwa,’ Takahiro Kanagawa’ and Satoshi Hanadal
Hiroyuki Hosoya,’
Author for correspondence : Tetsushi Suyama. Tel : + 8 1 298 54 659 1. Fax : + 8 1 298 54 6587.
e-mail: sutet@nibh.go.jp
1 National Institute of
Bioscience and Human
Technology, 1-1 Higashi,
Tsukuba, lbaraki
305-8566,
Japan
* Biological Laboratory,
Nippon Medical School,
Kawasaki 21 1-0063,
Japan
3.4 School of Dentistry3 and
Institute of Low
Temperature Science4,
Hokkaido University,
Sapporo 060, Japan
~~
Strains 61AT (T = type strain) and 6IB2, the first bacteriochlorophyll (BChI) a-
containing obligate aerobes to be classified in the /?-subclass of the
Profeobacteria, were isolated from river water. The strains were originally
isolated as degraders of poly(hexamethy1ene carbonate) (PHC). The organisms
can utilize PHC and some other biodegradable plastics. The strains grow only
under aerobic conditions. Good production of BChl a and carotenoid pigments
is achieved on PHC agar plates and an equivalent production is observed under
oligotrophic conditions on agar medium. Spectrometric results suggest that
BChl a is present in light-harvesting complex I and the photochemical reaction
centre. The main carotenoids are spirilloxanthin and its precursors. Analysis of
the 165 rRNA gene sequence indicated that the phylogenetic positions of the
two strains are similar to each other and that their closest relatives are the
genera Rubriwiwax, ldeonella and Leptothrix with similarities of 96.3, 962 and
96-1 % , respectively. The cells are motile, straight rods and contain poly-p-
hydroxybutyrate granules. Ubiquinone-8 is the predominant quinone. Vitamins
are not required for growth. The G+C content of genomic DNA is
66246.3 mol YO. Genetic and phenotypic features suggest that the strains
represent a new genus in the p-subclass which is evenly distant from known
genera. Consequently, the name Roseateles depolymerans gen. nov., sp. nov. is
proposed for the strains; the type strain of Roseateles depolymerans i s strain
61AT (= DSM 118133.
Keywords : aerobic photosynthetic bacteria, bacteriochlorophyll a, /3-subclass,
Proteobacteria, Roseateles depolymerans gen. nov., sp. nov.
INTRODUCTION
There are some species of bacteriochlorophyll (BChl)
a-containing bacteria that cannot grow photosyn-
thetically under anaerobic conditions even in light of
the effective wavelength. These organisms are referred
Abbreviations: BChl, bacteriochlorophyll; FD-MS, field desorption mass
spectrometry; LH-I, light-harvesting complex I; PHB, poly-0-hydroxybuty-
rate; PHC, poly(hexamethy1ene carbonate); UQ-8, ubiquinone with 8
isoprene units.
The GenBanUEMBL accession numbers for the 165 rRNA gene sequences of
strains 61AT and 61B2 are AB003623 and AB003625, respectively.
00785 0 1999 IUMS
to as aerobic photosynthetic bacteria (Harashima
et al., 1982; Shiba, 1989; Shimada, 1995). Although
the function of their photosynthetic apparatus has not
yet been clarified, light-stimulated growth (Harashima
et al., 1987; Yurkov & Gemerden, 1993) and photo-
synthetic activity (Harashima et al., 1982 ; Kortluke
et al., 1997; Okamura et al., 1986; Shiba, 1984;
Takamiya & Okamura, 1984) have been reported in
some species of aerobic photosynthetic bacteria.
Yurkov & Gemerden (1993) hypothesized an advan-
tage of having both photosynthetic and heterotrophic
metabolisms in terms of adaptation to the environment
with altering light/dark conditions under aerobic
449

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T. Suyama and others
conditions. Some physiological changes related to
adaptation to light/dark have been reported (Takaichi
et al., 1991; Tilden et al., 1997).
.Aerobic photosynthetic bacteria have been isolated
from various environments : freshwater (Fuerst et al.,
1993), marine cyanobacterial mat (Yurkov et al.,
1994), cyanobacterial mat from warm/hot spring
(Hanada et al., 1997; Yurkov & Gemerden, 1993;
Yurkov & Gorlenko, 1992, 1993; Yurkov et al., 1993)
and surfaces of seaweeds (Shiba, 1991; Shiba &
Simidu, 1982). Currently, aerobic photosynthetic bac-
teria are classified into the following genera : Erythro-
bacter and Roseobacter for marine species; and Por-
phyrobacter, Erythromicrobium and Roseococcus for
freshwater species. Recently, two genera, Sandara-
cinobacter and Erythromonas, were separated from
Erythromicrobium (Yurkov et al., 1997). Furthermore,
similar isolates have been reported from soil (Saitoh &
Nishimura, 1996), surfaces of plants (Urakami et al.,
1993), nodules of legumes (Evans et al., 1990), food,
animal feed and sewage sludge (Nishimura et al.,
1981), some of which are currently classified into
genera which have been considered as typical non-
phototrophs: Acidiphilium (possessing zinc-substi-
tuted BChl a> (Wakao et al., 1996), Bradyrhizobium
(Evans et al., 1990; Young et al., 1991) and Methylo-
bacterium (Green & Bousfield, 1983; Urakami et al.,
1993). So far, all of the genera described above have
been phylogenetically classified into the a-subclass of
the Proteobacteria. Five genera, Erythrobacter, Ery-
th romicr o bium, Ery t hr om onas, Porphyr obac ter and
Sandaracinobacter, belong to the a-4 group where
aerobic photosynthetic species are predominant and
the others belong to the a-1 to a-3 groups, which
contain close relatives of typical anoxygenic photo-
synthetic bacteria (Hanada et al., 1997; Shimada,
1995; Turova et al., 1995; Young et al., 1991 ; Yurkov
et al., 1994, 1997). No aerobic photosynthetic bac-
terium has been reported outside of the a-Proteo-
bacteria.
Two new obligate aerobes containing BChl a, strain
61AT (= DSM 11813T; T=type strain) and strain
61B2 (= DSM 1 1814), were isolated from river water
by screening aliphatic polycarbonate-degrading micro-
organisms (Suyama et al., 1998). Phylogenetic analysis
based on 16s rRNA comparisoiis revealed that these
strains belong to the p-subclass of the Proteobacteria;
the strains are thought to be the first aerobic photo-
synthetic bacteria found in this taxon.
In the present study, morphological, physiological,
biochemical and genetic characteristics of the strains
are described and a new genus name, Roseateles
depolymerans gen. nov., sp. nov., is proposed for the
isolates.
METHODS
Origin of strains. Strains 61AT and 61B2 were isolated from
the Hanamuro River in Tsukuba, Ibaraki Prefecture of
Japan. The strains were isolated with a medium containing
emulsified poly(hexamethy1ene carbonate) (PHC) as a sub-
strate at 30 "C (Suyama et al., 1998).
The type strain of Rubrivivax gelatinosus (DSM 1709T =
ATCC 1701 lT = NCIB 8290T = LMG 43 1 1') used in this
study as a reference species was purchased from Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ,
Braunschweig, Germany).
Culture media. The following media were used in the present
study. The agar plates were prepared by adding 1-5 % (w/v)
Bacto agar (Difco) to the media. PHC medium was used for
isolation and maintenance of original culture line. CAV
medium was used for the other physiological tests.
The PHC medium contained ( 1 3 1 g PHC (emulsified,
number-average molecular mass Mn = 2000; Toagosei) ; 1 g
(NH,),SO,; 0.1 g yeast extract (Difco) ; 20 mg CaC1,. 2H,O;
10 mg NaCl; 10 mg FeSO, . 7H,O ; 0.5 mg Na,MoO, . 2H,O;
0.5 mg Na,WO,. 2H,O; 0.5 mg MnSO, ; 60 mg surfactant
(Plysurf A210G; Daiichi Kogyo Seiyaku); 0.2 g KH,PO,;
and 1.6 g K,HPO, (pH 7-0).
The CAV medium contained (1-l): 2 g Casamino acids
(Difco); 0.5 g (NH,),SO,; 1 ml vitamin mixture; 10 ml basal
salt solution; and 25 ml 200 mM potassium phosphate
buffer (pH 7.0). The composition of the basal salt solution
and the vitamin mixture were previously described (Hanada
et al., 1997).
Physiological tests and determination of growth conditions.
The ability of the strains to grow photosynthetically under
anaerobic conditions was investigated both on the agar
plates and in liquid culture in the light. The anaerobic or
semi-aerobic growth on agar plates was assessed in an
anaerobic jar with Anaero Pack or Campylo Pack (Mitsu-
bishi Gas Chemical), respectively. The photoheterotrophic
growth was ascertained using PE medium (Hanada et al.,
1995b), which contains acetate, glutamate and succinate as
electron donors. The photoautotrophic growth in the pres-
ence of reduced sulfur compounds was determined in a
medium containing (1-I): 0.5 g (NH,),SO,;
S,O, .5H,O ; 0.1 g Na,S .9H,O ; 4.2 g NaHCO, ; 1 ml vitamin
mixture; 5 ml basal salt solution (Hanada et al., 1997); and
a final concentration of 10 mM potassium phosphate buffer
(pH 7.0).
The growth rates as a function of temperature were
determined in CAV medium. The vitamin requirement was
determined at 30 "C with special CAV medium lacking the
vitamin mixture and containing vitamin-free Casamino acids
(Difco). The final reading was obtained after nine serial
transfers. The growth rates at various pH were also
determined at 30 "C in a series of CAV media that were
buffered by 10 mM sodium citrate (pH 3-6), 10 mM pot-
assium phosphate (pH 7-8) or 10 mM sodium/glycine buffer
(PH 9-1 1).
0.5 g Na,
For the analysis of photosynthetic pigments, colonies
formed on PHC agar plate medium incubated for 3 d were
used. Pigments were extracted from cells with acetone/
methanol (7 : 2, v/v). The crude extract was analysed with an
HPLC system equipped with a p Bondapak C18 column
(8 x 100 mm; Waters) eluting with methanol (Takaichi &
Shimada, 1992). Major carotenoids were purified from silica
gel TLC (Merck) developed with n-hexane and acetone (4 : 1,
v/v). Absorption spectra were recorded with a photodiode
array detector (200-800 nm, MCPD-3600; Otsuka Elec-
tronics) attached to the HPLC apparatus (Takaichi &
Shimada, 1992). The molar absorption coefficients in meth-
anol (90.6 rnM-'cm-' at absorption maxima for carotenoids
450
International Journal of Systematic Bacteriology 49

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Roseateles depolyrnerans gen. nov., sp. nov.
and 54.9 mM-lcm-l at 770 nm for BChl a) were used for
quantification. The molecular masses were determined by
field desorption mass spectrometry (FD-MS), using a
double-focusing GC-MS equipped with field desorption
apparatus (M-2500 ; Hitachi) (Takaichi, 1993).
Quinones were extracted with chloroforrn/methanol (2 : 1,
v/v) from cells grown for 1 d in CAV medium. The extract
was purified by the method of Hiraishi et al. (1996) and
analysed by HPLC using a Gold HPLC System (Beckman)
with a ZORBAX-ODS column (Shimadzu).
The organic substrate utilization activities were determined
with BiOLOG GN and GP Microplates (Biolog) accord-
ing to the recommended protocol of the manufacturer.
Gelatinase activity was determined by the standard method
(Smibert & Krieg, 1981). Utilization of hydrogen gas was
investigated with 15 YO (v/v) hydrogen in air using carbon-
free medium at 30 "C.
Nitrogen-fixing ability was tested by acetylene reduction
assay (Inoue et al., 1996). Overnight culture was prepared in
a medium that contained (1-I): 5 g glucose; 1 g Agar Noble
(Difco); 10 ml basal salt solution; and 25 ml 200 mM
potassium phosphate buffer (pH 7.0). The culture was placed
in a rubber-capped tube with 15% (v/v) acetylene and
incubated at 30 "C. Reduction of acetylene was assayed with
a GC-8A gas chromatograph (Shimadzu) with a Unibeads C
60/80 column (GL Science).
Electron microscopy. Negatively stained cells and ultrathin
sections of the cells were observed with a model H-7000
electron microscope operating at 75 kV (Hitachi). The cells
were collected from PHC agar plates (3 d) or from CAV
liquid medium (1 d). Cells on a collodion membrane were
negatively stained with 1 YO (w/v) aqueous uranyl acetate.
Ultrathin sections were prepared by a previously described
method (Hanada et al., 1995a). The cells were suspended in
Kellenberger buffer (Kellenberger et al., 1958) and em-
bedded in 1 YO (w/v) agar. The specimen was fixed with 2.5 YO
(w/v) glutaraldehyde and 1 YO (w/v) OsO,, prestained with
ruthenium red and uranyl acetate, and embedded in epoxy
resin.
DNA composition and phylogenetic analysis. Genomic DNA
was extracted and purified by the method of Marmur (1961).
Almost full-length 16s rRNA genes were amplified by PCR
with a pair of primers designed from positions 8-27 and
1492-1 5 1 1 of the Escherichia coli 16s rRNA gene (Brosius et
al., 1978; Weisburg et al., 1991). The PCR products cloned
into plasmid vector (pT7Blue T-Vector ; Novagen) were
sequenced separately by an automated sequencer (ABI
model 373A; Applied Biosystems) from both fonvard- and
reverse-strands using twelve kinds of primers. The detailed
procedure for the sequencing was described previously
(Suyama et al., 1998).
The G+C content was determined by the protocol of
Kamagata & Mikami (1991).
Nucleotide sequence accession numbers. Accession numbers
for the reference 16s rRNA gene sequences were as follows :
Alcaligenes latus, D88007 ; Brachymonas denitrijicans,
D 14320; Comamonas testosteroni, M 1 1224; Ideonella dech-
loratans, X72724 ; Leptothrix cholodnii, X97070 ; Leptothrix
discophora, L33974; Leptothrix mobilis, X9707 1 ; Polaro-
monas vacuolata, U 14585 ; Rhodocyclus tenuis, D 16208 ;
Rhodoferax fermentans, D 162 I2 ; Rubrivivax gelatinosus,
D16213 ; Sphaerotilus natans, Z18534; Thiobacillus thermo-
sulfatus, U27839 ; and Variovorax paradoxus, D30793.
RESULTS
Strains and growth conditions
Two strains, 61AT (soft and smooth colony) and 61B2
(harder and larger colony than that of 61AT), were
isolated from the Hanamuro River (Ibaraki Prefecture,
Japan) as two PHC-degrading strains ; the strains co-
metabolically utilized PHC (Suyama et al., 1998). Both
of the strains formed pink colonies on PHC agar plates
within 2-3 d, making clear zones caused by degra-
dation of PHC around their colonies. Other biode-
gradable plastics, poly(e-caprolactone) and poly(tetra-
methylene carbonate) (Suyama & Tokiwa, 1997), were
degraded in the same way.
The strains were able to grow in CAV medium buffered
at pH 5-8 (Fig. la). Growth was observed at 5-43 "C,
and an incubation temperature of 45 "C was lethal
(Fig. lb). Optimal pH and temperature for the growth
of these strains were pH 6.5 and 35 "C, respectively.
No vitamins were required for growth; subculturing
strains 61AT and 61B2 in CAV medium lacking
vitamins had no effect on growth.
Both of the strains produced a musty odour and
mucus. Strain 61B2 tended to make white floc in liquid
culture whereas strain 61AT did not,
0.6 I
Temperature ("C)
................................................................................................................................. ...... .......... ...
Fig. 7, Growth rate of strains 61AT (0) and 61B2 (0)
function of pH at 30 "C (a) and as a function of temperature at
pH 7.0 (b). Cells were grown in CAV medium under aerobic
conditions in the dark.
as a
International Journal of Systematic Bacteriology 49
45 1

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T. Suyama and others
500 600 700 800 900
Wavelength (nm)
500 600
Wavelength (nm)
700
800
900
. . . . . . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig- 2. Absorption spectra of (a) intact cells of strain 61AT
grown on PHC agar plates (solid line) and CAV agar plates
(dashed line) for 5d; and (b) ultrasonically disrupted
membrane in potassium phosphate buffer, pH 6.8 (solid line),
and acetone/methanol (7:2, v/v) extract (dotted line) of the
cells grown on PHC agar plates. Spectra were recorded with a
Beckman DU-640 spectrophotometer.
Photosynthetic pigments
The strains grew on agar plates of CAV, nutrient broth
or 0.1 % skim milk media, but the colonies were
colourless. When the strains were grown on agar plates
with a lower concentration of carbon sources (e.g. in
the presence of 0.02% Casamino acids), weak pig-
mentation was observed. In vivo absorption spectra of
intact cells of strain 61AT (pigmented and non-
pigmented) are shown in Fig. 2(a).The cells grown on
PHC agar plates showed a large absorption peak at
870nm, whereas those grown on CAV agar plates
showed no significant peak around this wavelength.
No pigmentation was observed in liquid-cultured cells.
The strains did not grow anaerobically and photo-
synthetically under conditions used for purple sulfur
or non-sulfur bacteria. Growth and the production of
pigments by the strains were blocked under anaerobic
conditions. The strains were able to grow and pro-
duced the pigments in semi-aerobic conditions
(&10?40, v/v, 0,).
At present, the PHC agar plate is the best medium for
pigment production by strains 6 1 AT and 6 1 B2. Greater
pigment production was observed in dark-grown cells
compared to light-grown cells. Ultrasonically dis-
rupted cells had absorption maxima at 482, 515, 550,
590, 800 and 870 nm (solid line; Fig. 2b). The
Fig, 3 . Electron micrographs of negatively stained cells of strain
61AT grown in CAV medium for 1 d (a) and ultrathin cross-
sections of cells of strain 61AT grown on a PHC agar plate for
3 d (b). The outer membrane (OM), cytoplasmic membrane (CM)
and intracellular granules (G) are shown in (b). Cells were post-
stained with uranyl acetate and lead citrate. No invaginations
of intracytoplasmic membranes were observed. Bar, 1 pm.
absorption peaks of the pigments extracted with
acetone/methanol(7 : 2, v/v) were shifted as indicated
in the figure (dotted line; Fig. 2b). BChl a with phytol
ester was confirmed by the retention time on HPLC
and absorption spectrum. Analysis of carotenoids
using HPLC and FD-MS revealed that the major
carotenoid of these strains was spirilloxanthin (M,
596; 89 and 88 mol% of total carotenoids in strains
61AT and 61B2, respectively). The precursors of
spirilloxanthin, i.e. OH-spirilloxanthin (2 and
3 mol '?LO, respectively), anhydrorhodovibrin (8 and
7 mol YO) and 3,4-dehydrorhodopin (1 and 2 mol YO),
were also detected as minor components. The molar
ratios of total carotenoids/BChl a were 0-65 (strain
61AT) and 0.58 (strain 61B2). Spheroidene (or sphero-
452
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Roseateles depolymerans gen. nov., sp. nov.
idenone), which is one of the major carotenoids of
Rubrivivax gelatinosus, the phylogenetic neighbour
described below, was not detected.
The production of photosynthetic pigments in Rubri-
vivax gelatinosus cells was determined under the same
conditions as the two strains using CAV or PHC
medium. Rubrivivax gelatinosus produced BChl a and
carotenoids in liquid CAV medium or on CAV agar
plates. However, pigment production was not ob-
served on PHC agar plates.
Morphology and ultrastructure
Strains 61AT and 61B2 had similar morphological
characteristics. Cells of strain 61AT grown in CAV
medium were motile (motility was observed only in the
early exponential phase in a liquid culture), Gram-
negative rods that were 0.5 x 2 pm with flagella (Fig.
3a). No intracytoplasmic membranes were observed in
ultrathin sections of pigmented cells of strain 61AT
(Fig. 3b). Intracellular granules were observed, some
of which were considered to be identical to the poly-P-
hydroxybutyrate (PHB) granules determined by Nile
Blue A staining (Suyama et al., 1998). No remarkable
differences were observed in ultrathin sections between
the cells with and without photosynthetic pigments.
Phylogenetic analysis
The obtained sequence of the 16s rRNA genes of each
isolate consisted of 1452 bp. The sequences of strains
61AT and 6 1 B2 were identical except for a single base
(i.e. guanine at E. coli position 648 in strain 61AT was
replaced by adenine in strain 61B2).
Fig. 4 shows the phylogenetic positions of the strains
among representative members of the P-subclass of the
Proteobacteriu, determined by the neighbour-joining
method (Saitou & Nei, 1987). The sequence of Rhodo-
cyclus tenuis was used as the outgroup reference.
Alcaligenes latus, Ideonella dechloratans, Rubrivivax
gelatinosus, Sphaerotilus natans and the Leptothrix
species were the phylogenetic neighbours of strains
61AT and 61B2. Rubrivivax gelatinosus was the only
phototrophic species among closely related organisms.
The closest relatives of the two strains were Rubrivivax
gelatinosus, Ideonella dechloratans, Leptothrix cholod-
nii and Leptothrix discophoru with similarities of 96.3,
96.2, 96.1 and 96-1 %, respectively.
Physiological and other properties
The results of the nutritional and biochemical tests
carried out on strains 61AT and 61B2 are summarized
in Table 1. The reference data for the phylogenetic
neighbours are listed together. The strains could utilize
the organic acids and carbohydrates listed in Table 1 as
sole carbon sources. The strains utilized organic acids
(e.g. L-malate, succinate, etc.) and the reducing mono-
hexoses (e.g. glucose, fructose, etc.) well. Some other
physiological traits of the two strains were reported
previously (Suyama et al., 1998). The major quinone
Thiobacillus thermosundlus I
Polaromonas vocuolata
Rhodocyclus tanuls oYq
Brxhymonas dsnltrificms
Comamonas tsstosteroni
Vadovorax peradoxus
Rhodofarax termantans
strain 6182
Ideonella dachloratans
7
Rubriviwx galrtlnosus
I
Leptothrix mobilis
Lcptothdx cholodnii
Laptothrix discophora
I
Sphaamtllus natans
0.01
Fig. 4. Phylogenetic positions of strains 61AT and 61B2 among
neighbouring species selected from the P-subclass of the
Proteobacteria. Bar,
1
nucleotide substitution per
nucleotides in 165 rRNA gene sequences. The numbers at the
nodes of the tree indicate bootstrap values (%) for each node
of 1000 bootstrap resamplings.
100
component of strains 61AT and 61B2 was UQ-8
(ubiquinone with 8 isoprene units). The G + C contents
of the strains, determined by HPLC, were 66-3 (strain
61AT) and 66.2 (strain 61B2) mol %.
DISCUSSION
Strains 6 1 AT and 6 1 B2 were characterized previously
as physiologically new strains that co-metabolically
utilized aliphatic polycarbonates (Suyama et al., 1998).
The two strains grew only under aerobic conditions
with production of BChl a. Light did not support
growth of the strains under anaerobic conditions.
These features are similar to those of aerobic photo-
synthetic bacteria belonging to the a-subclass of the
Proteobacteria (Evans et al., 1990 ; Fuerst et al., 1993 ;
Green & Bousfield, 1983; Shiba, 1991; Shiba &
Simidu, 1982; Urakami et al., 1993; Wakao et al.,
1996; Young et al., 1991; Yurkov et al., 1993, 1997;
Yurkov & Gorlenko, 1992). The absence of intracyto-
plasmic membranes as shown in Fig. 3(b) has also
been reported in some other aerobic photosynthetic
bacteria (Hanada et al., 1997; Yurkov et al., 1994).
The 16s rRNA gene sequences indicate that the strains
are distinguishable from the a-subclass of the Proteo-
bacteria and that they belong to the P-subclass. The
predominant presence of UQ-8 in the isolates as the
major quinone component supports their taxonomic
position in the P-subclass of the Proteobacteria (Hira-
ishi et al., 1996).
Absorption maxima at 870 and 800 nm in Fig. 2(b)
indicate the presence of BChl a in the light-harvesting
complex I (LH-I) and the photochemical reaction
International Journal of Systematic Bacteriology 49
453

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T. Suyama and others
Table 1. Characteristics of the species phylogenetically close to strains 61AT and 61 B2
+ , 2 90 YO positive; - , < 10 % positive; d, 11-89 YO of the strains positive; NT, not tested. The following references were used:
Imhoff & Triiper (1989); Kersters & De Ley (1984); Malmqvist et al. (1994); Suyama et al. (1998); and Willems et al. (1991).
1, Strains 61AT and 61 B2; 2, Ideonella dechloratans; 3, Rubrivivax gelatinosus; 4, Alcaligenes latus; 5, Leptothrix discophora; and
6, Sphaerotilus natans.
...................................................................................................................................................... .........................................................................................................................................................
~~
Characteristic
1
2
3
4
5
6
Motility
Flagellation
Cytochrome c oxidase
Catalase
Formation of sheaths
Phototrophc growth
Autotrophic growth with hydrogen
Nitrogen fixation
Nitrate reduction
Gelatinase
Accumulation of PHB
Pigments:
BChl a
Carotenoids
Growth factors required
Carbon sources utilized :
Acetate
Pyruvate
Lactate
L-Malate
Succinate
Citrate
D-Ribose
D-Glucose
D-Fructose
D-Galactose
Sucrose
Glycerol
Mannitol
Sorbitol
Isolation source
G + C content (mol YO)
* The pigments are produced by cells grown aerobically on PHC agar medium in the dark.
The pigments are produced by cells grown anaerobically in the light.
+
+
-
-
-
-
-
-
+
+
+*
Several, polar
Spirilloxanthin*
-
d
+
+
+
+
+
-
+
+
+
-
-
+
-
Water
66'2-66'3
+
+
+
-
-
Several, polar
NT
NT
+(or chlorate)
NT
NT
-
-
+I
+
+
+
NT
+
NT
NT
+
+
NT
NT
NT
NT
NT
Water
68.1
+
1, polar
NT
NT
-
+
+
+
NT
+
NT
+'r
Spheroidene,
OH-spheroidene,
spirilloxanthint
Biotin, thiamin
+
+
+
+
+
+
NT
+
+
NT
NT
-
-
-
Mud
70.s72.5
+
+
NT
-
-
+
+
-
+
+
5-10, peritrichous
NT
-
-
+
+
+
d
-
+
+
-
+
+
-
-
Soil
69.1-7 1.1
+
1, polar
NT
NT
+
-
-
NT
NT
NT
+
Vitamin B,,
-
+
-
+
+
-
-
+(cannot grow)
-
NT
+(not all strains)
+
NT
NT
Water
67.8-7 1.1
+
+
+
+
-
Polar bunch
NT
NT
NT
+
+
-
-
Vitamin B,,
+
+
+
+
+
+
NT
+
+
+
+
+
+
+
Water
69.9
$ See reference by Malmqvist et al. (1994).
centre. In the pigmented cells, the molar ratio of
carotenoids/BChl a in the strains was always about
0.6. This stoichiometry is quite similar to the LH-I in
Rhodospirillum rubrum (Cogdell et al., 1982), and a
major proportion of the carotenoids is considered to
be bound to the complex. Although most of the species
of aerobic photosynthetic bacteria are known to
contain carotenoids which do not bind to the photo-
synthetic apparatus (e.g. erythroxanthin sulfate), such
components were not detected in strains 61AT and
61B2.
The species listed in Table 1 are the phylogenetic
neighbours of the strains suggested by analysis based
on the 16s rRNA gene sequences (Fig. 4). The presence
of motility, flagellation, PHB granules, oxidase and
gelatinase are common characteristics of the species in
this taxon. The absence of sheaths clearly distinguishes
the strains from the genera Sphaerotilus and Lepto-
thrix. The difference in the utilization pattern of carbon
sources (Table 1) suggests that the strains are distinct
from known species. The G+C contents of the two
454
strains are comparatively low in the phylogenetic
cluster and distinguish the strains from relatives in this
taxon. The formation of photosynthetic pigments is
the most remarkable characteristic of strains 61AT and
61 B2. Although Rubrivivax gelatinosus is the only
species containing BChl a among the phylogenetic
neighbours, the growth physiology and the production
of pigments in Rubrivivax gelatinosus were different
from those of the two strains. Rubrivivax gelatinosus is
a typical anoxygenic phototrophic bacteria and grows
under both aerobic and anaerobic conditions. Rubri-
vivax gelatinosus has the spheroidene and spirillo-
xanthin series as major carotenoid species while the
two strains have only the spirilloxanthin series. Ni-
trogen fixation and autotrophic growth on hydrogen
were reported for Rubrivivax gelatinosus (Imhoff &
Truper, 1989), but were not found in the two strains.
Rubrivivax gelatinosus requires biotin and thiamin for
growth, whereas the strains could grow in a vitamin-
free medium.
The 16s rRNA gene sequence similarities to the
International Journal of Systematic Bacteriology 49

Page 7

Roseateles depolqwerans gen. nov., sp. nov.
~~
phylogenetic neighbours, Rubrivivax gelatinosus, Ideo-
nella dechloratans and the Leptothrix species, were
96.3, 96.2 and 96-1 YO, respectively. The distances to
each of the genera are sufficient to consider creation of
a new genus. The bootstrap probabilities indicated in
Fig. 4 show that the genera around strains 61AT and
61B2 are equally dispersed under the branch with a
reliability of 100 YO. Genetic and phenotypic features
suggest that strains 61AT and 61B2 represent a new
genus in the P-subclass of the Proteobacteria which is
evenly distant from known genera. The name Rose-
ateles depolymerans gen. nov., sp. nov. is therefore
proposed for the strains.
Description of Roseateles gen. nov.
Roseateles (ro.se.a.te'les. L. adj. ruseus rose-coloured,
pink; Gr. adj. ateles defective, incomplete; N.L. masc.
n. roseateles the rose-coloured incomplete photosyn-
thetic bacterium).
Cells are motile, Gram-negative straight rods. Cells
possess PHB granules as a storage material and
reproduce by binary fission. The bacterium grows
heterotrophically under aerobic conditions. BChl a
with phytol ester and carotenoid pigments are formed.
Colonies are pink in colour under conditions suitable
for photosynthetic pigment production. The bacterium
cannot grow anaerobically even in the light. The major
quinone is UQ-8. On the basis of the results of a 16s
rRNA gene sequence comparison, the bacteria belong
to the P-subclass of the Proteohacteria. DNA G+C
content is 66-2-66.3 mol %. The type species is Rose-
ateles depolymerans.
Description of Roseateles depolymerans sp. nov.
Roseateles depolymerans (de. po .ly'me.rans. N. L. v.
depolymerare depolymerize ; N.L. part. adj. depoly-
merans depolymerizing) .
Characteristics are the same as those given in the
description of the genus. Lives in river water. BChl a
with phytol ester and spirilloxanthin are present along
with the LH-I when cells are grown aerobically on agar
plates supplemented with low levels of carbon sources,
e.g. 0.02 YO Casamino acids. Grows well heterotro-
phically with rich media containing 0.2 % Casamino
acids, 0-8 Yo nutrient broth or 0-1 % skim milk, under
aerobic conditions. In this case, BChl a and caro-
tenoids are hardly produced. Hydrogen is not utilized
for autotrophic growth. Positive for gelatinase and
oxidase. Negative for catalase, nitrogen fixation, ni-
trogen reduction and denitrification. No vitamins are
required for growth. Optimum pH for the growth is
pH 6-5 and optimum temperature is 35 "C. The culture
is not maintained at 45 "C. The bacterium can grow on
D-glucose, D-fructose, D-galactose, mannitol, pyru-
vate, lactate, L-malate, succinate, citrate, Casamino
acids or yeast extract as a sole carbon source. Degrades
PHC, poly(tetramethy1ene carbonate) and poly(e-
caprolactone) by means of co-metabolism. Type strain
is 61AT, which has been deposited in the Deutsche
Sammlung von Mikroorganismen und Zellkulturen as
DSM 11813T.
ACKNOWLEDGEMENTS
We wish to thank Hans G. Truper (Universitat Bonn) for
advice and correction of the Latin names.
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